Medical Hypotheses (2003) 60(4), 520–524

ª 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0306-9877(02)00451-6

Induction of thymic tolerance

as possibility in prevention
of recurrent spontaneous abortion
I. V. Bubanovic
Department of Obstetretics and Gynecology – Health Center in Gnjilane, Yugoslavia

Summary A major process through which the immune system becomes tolerant to self-proteins involves the
deletion of self-reactive cells in the thymus and/or inhibition of specific Th1 cells clones. Deletion process includes
two selection mechanisms in which the thymus eliminates unwanted thymocytes are known as positive selection
and negative selection. The thymus is an antigenically privileged site, mainly for it is discrete by blood-thymus
barrier. Many researches were shown that intrathymic inoculation of any antigen resulted in specific tolerance
induction. The embryo/fetus and placenta are an allograft to which the mother must remain immunologically
tolerant in order for the fetus to survive. Today, there is much interest focused on the immunology of recurrent
spontaneous abortion (RSA). Up to 50% of RSA may be mediated by the immune system via inadequate maternal
anti-paternal response. Nature of this maternal–fetal disturbance represents disbalance in Th1 =Th2 activity.
Contra-shift in Th1 =Th2 activity is the basis for immunotherapy with paternal leukocyte immunization (PLI). PLI
induce some kind of peripheral tolerance on embryonic/fetal/trophoblast antigens, but problems of central
tolerance are still open. Intrathymic inoculation of fetal or paternal cells (like leukocyte, thymic dendritic cells,
trophoblast cells) or paternal set of MHC molecules may cause central specific tolerance and may be a new
possibility for immunotherapy in RSA patients.
ª 2003 Elsevier Science Ltd. All rights reserved.

BASIC MECHANISMS OF THYMIC TOLERANCE usual cell types like the rhabdomyocyte, which appear
to represent antigenic stereotypes of normal host cells.
During embryonic development of every mammals,
The epithelial cells are very specific regarding special
their immune system undergoing phase of ‘education’ to
structure and expression of antigenic markers. Interac-
recognize self and nonself structure. In most cases, this
tion of precursor T-cells with these various stromal an-
process happens in embryonic or early fetal phase, but
tigens and antigens presenting cells probably
some species like rodents can finish ‘education’ in early
determinate whether immunoreactivity or tolerance re-
postnatal phase. Only immature T lymphocytes are lia-
sults (2,3).
ble to be processed across ‘education’ in the thymus (1).
The two selection processes in which the thymus
Development and maturation of T-cells in the thymus
eliminates unwanted thymocytes are known as positive
is under the influence of the epithelial and mesenchymal
selection and negative selection. Thymic stromal cells,
stromal cells. Cells of mesenchymal stroma include
including epithelial cells, macrophages and dendritic
mononuclear phagocytes, Langerhans-like cells and un-
cells, are thought to play a role in positive and negative
selection. Thymic stromal cells express high levels of
class I and class II MHC molecules allowing immature
Received 4 April 2002
thymocytes expressing the TcR–CD3 complex to inter-
Accepted 11 July 2002
act, leading to positive and negative selection (1,4–6).
Positive selection involves an interaction of immature
Correspondence to: I.V. Bubanovic, Department of Obstetretics and
Gynecology – Health Center in Gnjilane, Yugoslavia. thymocytes with epithelial cells in the cortex of the
E-mail: ibubanovic@yahoo.com thymus. During positive selection, the RAG-1, RAG-2

520

and TdT proteins continue to be expressed. The imma-
ture thymocytes in a clone expressing a given b chain
continue to rearrange TcR a chain genes. The resulting
TcR’s are then selected for s-MHC recognition. Only
those cells whose ab-TcR heterodimer recognizes a self-
MHC molecule are selected for survival. Interaction of
immature CD4þ 8þ thymocytes bearing receptors capa-
ble of binding self-MHC molecules, with thymic epi-
thelial cells survive as they may receive a protective
signal on binding. Those cells whose receptors are not
MHC restricted do not interact with thymic epithelial
cells and consequently do not receive the protective
signal, thus leading to their death within 3–4 days via
apoptosis (1,4–6).
Negative selection involves the population of MHC-
restricted thymocytes that survive positive selection
comprises of some cells with low affinity receptors for
self-antigen presented by self-MHC molecules and other
cells with high affinity receptors. The latter thymocytes
undergo negative selection by an interaction with bone
marrow derived APCs (dendritic cells and macrophages)
in the medulla. During negative selection, dendritic cells
and macrophages bearing class I and class II MHC
molecules are thought to interact with thymocytes
bearing high affinity receptors for self-antigen plus self-
MHC molecules, or self-MHC molecules alone. The na-
ture of the interaction is unknown, but the selected cells
undergo death by apoptosis (1,3,5). Apoptotic selection
of specific clones of thymocytes is not single mecha-
nisms of thymic tolerance induction. Some authors were
found that thymic antigen expression results in anergic
thymocytes, which also show a reduction of Th1 activity
with no decrease in Th2 activity (2). This mechanism
may be important for restitution of Th2 domination in
pregnancy and in tumor bearers.
None of thymic epithelial cells express class II MHC
antigen. The subcapsular epithelial lamination is MHC
class II negative, but the remaining cortex, including
the thymic nurse cells, is strongly MHC class II positive.
All cells in the thymus express MHC class I molecules
but the highest expression is on nonepithelial cells. Al-
though self/nonself ‘education’ and acquisition of hap-
lotype restriction of developing T cells requires
intrathymic expressed MHC molecules (1,3,6).
Studies with tracers of blood vessel permeability
showed that cortical thymocytes are protected from
circulating macromolecules. In contrast, medullar thy-
mic lymphocytes are relative unprotected from circu-
lating macromolecules. Blood vessels in the thymic
cortex were not permeable to the protein tracers, but the
postcapillary venules of the thymic medulla permitted
traces to leak along the clefts between migrating lym-
phocytes and endothelial cells. The tracers, which cros-
sed medullary venule walls, had limited distribution in
ª 2003 Elsevier Science Ltd. All rights reserved.
Induction of thymic tolerance 521
the thymic parenchyma because macrophages in the
perivascular space ingest and retain much of the leaked
tracer. The phagocytic cells and basal lamina at the
border between the cortex and medulla captured any
tracer that may have escaped endocytosis in the vessel
wall. Thus, hematogenous antigen has access, albeit re-
stricted, to the thymic medulla (7–9).
The thymus is widely thought to be an antigenically
privileged site, yet antigen is essential for positive selec-
tion of specific T cell clones for maturation and release to
the periphery, acquisition of MHC haplotype restriction
and tolerance induction implies that antigen must be
present. A so-called blood-thymus barrier was proposed
because of differences in ex vivo antibody formation in
thymus fragments after injecting antigen directly into
the thymus or after intravenous inoculation (1,2,10).
Other scientists used a different antigen but a similar
experimental design and found that intrathymic inocu-
lation with antigen resulted in the induction of specific
tolerance.
Every molecule that is presented in developing the
thymus induces specific thymic immunotolerance in
postnatal life (4). The restitution of thymic tolerance in
prenatal period is possible loading antigen with subcu-
taneous or intraperitoneal injection. In adult stage, the
restitution of thymic tolerance is possible only with in-
trathymic antigen inoculation, based on reeducation of
immature thymic lymphocytes (11). Most important
reason for this relationship is in nature of blood-thymus
barrier. Namely, blood-thymus barrier in embryonic
stage is permeable for most antigens, but adult thymus
have compact and impermeable blood-thymus barrier.
Impermeability of blood-thymus barrier in adult stage is
important for prevention of thymic tolerance to mi-
crobes and tumors.
Specific tolerance after intrathymic inoculation of
antigens is obtained in many experimental models and
different experimental condition. Induction of neonatal
thymic tolerance in newborn mice to foreign MHC
molecules was given by intravenous injection of alloge-
neic peritoneal or thymic cells (12,13). Many authors
used intrathymic inoculation of antigens for prevention
some autoimmune disease. Intrathymic injection of
guinea pig myelin basic protein without otherwise
compromising the peripheral lymphocyte pool in adult
rats dramatically inhibits onset of experimental allergic
encephalomyelitis caused by the usual peripheral inoc-
ulation with in complete Freund’s adjuvant (14,15).
Najman et al. (16) inoculated thymic epithelial cells of
a donor into the thymus of a recipient of the allogeneic
skin graft. Adult animals, recipients of donor’s thymic
epithelial cells and donors allogeneic the skin graft,
significantly slower reject the skin graft in comparison
with control animals. These authors instigate that
Medical Hypotheses (2003) 60(4), 520–524
522 Bubanovic
intrathymic inoculation of epithelial cells from donor to
recipient induce specific thymic tolerance to allogeneic
skin transplant. This is the first demonstration that in-
trathymic injection of allogeneic epithelial thymic cells
can induce acquired thymic tolerance (16).
Garrovillo et al. (17) instigate that intrathymic injec-
tion of an immunodominant peptide induces acquired
thymic tolerance suggests an indirect pathway of allo-
recognition in the thymus. They are found that geneti-
cally engineered dendritic cells expressing donor MHC
class I or II molecules or a peptide analogue might have
therapeutic potential in the induction of transplant tol-
erance and in the treatment of autoimmune diseases
(17). Similar finding was given with intrathymic injection
of UV-B-irradiated spleen cells or purified resting allo-
geneic T cells, but not resting B cells, dendritic cells, or
macrophages. Thymectomy performed 7 days after in-
trathymic injection led to graft rejection strongly sug-
gests that the early phase of induction of donor-specific
tolerance is dependent on the presence of donor al-
loantigens in the host thymus (18).
BASIC MECHANISMS OF RECURRENT
SPONTANEOUS ABORTION MEDIATED BY
IMMUNE SYSTEM AND PLI PROCEDURE
Central question of reproductive immunology is mech-
anisms of embryonic/fetal surviving in maternal MHC
incompatible environment. Most important barrier that
locks out direct contact between maternal immune sys-
tem and embryonic/fetal tissue is placenta. For many
years the placenta was thought to be nonimmunogenic
as it does not express class II MHC molecules, and class I
MHC expression is restricted to certain trophoblast cells.
Neither class I nor II MHC molecules are expressed on
the surface of human trophoblast in intimate contact
with maternal blood (19), except nonclassical MHC class
I molecules HLA-G, HLA-C and HLA-E (20). Expression
of HLA-G may be beneficial for pregnancy. Allogeneic
stimulator cells, when transfected with HLA-G gene,
promote cytokine pattern beneficial for pregnancy (21),
and HLA-G expression has been associated with in-
creased resistance to lysis by NK cells (22). However, in
especially conditions, expression of MHC molecules on
trophoblast cells may be exalted. These conditions im-
plies acceleration of Th1 activity, domination of cyto-
kines like IL-2, TNF-a, IFN-c, IL-12, IL-18 and activation
of CTL and NK cells cascade. Infective agents, primary
MHC expression on trophoblast cells, poor trophoblast
signals, mistake in decidual response on trophoblast
signals are most frequent reason for activation of Th1
cascade and immune mediated abortion. In any case, the
immune causes for pregnancy loss and implantation
failure are the result of abnormalities in balance between
Medical Hypotheses (2003) 60(4), 520–524
Th1 and Th2 activity of immune response, as reflex of the
immune intolerance. Th1 cytokines and activated NK
and CTL induce trophoblast cells lysis or apoptosis
(23,24).
Down-regulation of Th1 activity in account of Th2
type has been demonstrated in normal pregnancy (25)
prompting the hypothesis that successful pregnancy is
a Th2 phenomenon (26). Cytokine network of Th2 type
response include IL-4, IL-5, IL-6, IL-10 and probably
TGF-b. These cytokines are most important so-called
embryo-protective factors.In abortion prone combina-
tion (CBA/JxDBA/2J) the expression of mRNA for IL-2,
IFN-c and TNF-a were reported to be quantitatively
higher in the placentae of mice undergoing resorption
compared to placentae from normal murine pregnan-
cies (27). Levels of IL-2 and IFN-c were also increased
in supernatants of mixed lymphocytes and placental
cells from this same animal model of spontaneous
abortion (28). NK cells from decidua of normal preg-
nancy are cytotoxic only for some tumors lines, but
NK cells from decidua of abortion prone mice are cy-
totoxic for most tumor lines and trophoblast cells
(22,23,29).
Similar results were given in researches on women
with unexplained RSA. It is assumed that 80% of RSA of
unknown etiology are of immunological origin. The
proposed experimental treatment of those patients could
be immunization with paternal lymphocytes, though the
exact mechanism through which this therapy exerts its
effect is still unknown. Many authors suggest that lym-
phocyte alloimmunization alters the number and activ-
ity of particular subpopulations of peripheral blood and
decidual lymphocytes and/or modulate of immunity in
women with unexplained RSA by contra-shift in the
balance of cytokine profiles away from Th1 to a Th2
profile after immunotherapy (19,22). In research of Ha-
yakawa et al. (30) seven of nine patients who exhibited
remarkable decreases in Th1 /Th2 activity ratios became
pregnant within 6 months after three courses of immu-
notherapy, and four women delivered healthy babies,
while none of the three patients who exhibited an in-
creased or unchanged Th1 /Th2 activity ratio had full-
term pregnancies (30). PLI efficiently suppresses NK-cell
cytotoxicity and CD56+ NK-cell levels and increases the
peripheral blood CD3+ T-cell population in women with
recurrent spontaneous abortions (31). Moreover, PLI
significantly increase decidual production of IL-4, IL-6,
IL-10 and TGF-b and decrease production of IL-2, TNF-a
and IFN-c. In some cases, PLI cannot move Th1 =Th2
balance in account of Th2 activity, then RSA persist.
Many researches have shown efficiency of PLI in pre-
vention of RSA. Most important mechanisms of PLI are
in acceleration of Th2 and suppression of Th1 activity
(19,23).
ª 2003 Elsevier Science Ltd. All rights reserved.

Complications after PLI may be manifested as maternal
cutaneous graft-versus-host-like reaction (32), alloim-
mune neonatal neutropenia (33), neonatal thrombocyto-
penia (34), or perplexity in perinatal and neonatal
adaptation (35).
EXPERIMENTAL MODELS AND INTEREST FOR
IMMUNOTHERAPY OF RSA
Although thymic tolerance is efficient in experimental
models like prevention of autoimmune disease or allo-
graft rejection, hypothesis that thymic tolerance maybe
useful in prevention of RSA is real.
Possibility that leucocytes injected with PLI proce-
dure populate the thymus of recipient and induce tol-
erance and/or suppression of Th1 activity is also real. It
can be presumably explanation for efficiency of PLI
procedure.
The intrathymic injection of paternal lymphocytes
into maternal thymus with or without intravenous PLI
may cause central and peripheral immune tolerance.
Because paternal lymphocytes may be dangerous for
maternal thymus, paternal MHC molecules expressed
on liposome membrane may be inoculated in maternal
thymus with or without liposome intravenous injec-
tion.
Most elegant, but in same time most complicated
models is intrathymic inoculation of paternal or fetal
thymic epithelial and dendritic cells into maternal thy-
mus.
Fetal/embryonic cells or antigens may be utilized in
same destination as paternal cells or antigens.
Most improvident results are expected in case of in-
trathymic inoculation of trophoblast cells or antigens.
Trophoblast cells from previous aborted pregnancy or
received by biopsy procedure during pregnancy may be
used for inoculation into maternal thymus.
Good experimental model for induction of thymic
tolerance in pregnancy is abortion prone (CBA/Jx DBA/
2J) combination of mice. Intravenous treatment of
CBA/J female with DBA/2J lymphocytes is inefficient,
but treatment with BALB/c lymphocytes is very effi-
cient in prevention of embryo resorption. Inoculation
of immortalized DBA/2J lymphocytes or trophoblast
cells into the thymus of CBA/J female may cause better
results in prevention of embryo resorption.
Inoculation of paternal cells or antigens into maternal
thymus may provide deletion of specific antipaternal
clone of lymphocytes and may provide suppression of
Th1 activity along development of Th2 activity.
As results of these events, procedure of thymic tol-
erance induction may be used in prevention or treat-
ment of RSA syndrome.
ª 2003 Elsevier Science Ltd. All rights reserved.
Induction of thymic tolerance 523
Except RSA, intrathymic tolerance may be useful in
treatment of preeclampsia, PIH syndrome, or in pre-
vention of PLI procedure complications.
REFERENCES
1. Nossal G. J. V. Negative selection of lymphocytes. Cell 1994;
76: 229–240.
2. Antonia S. J., Geiger T., Miller J., Flavell R. A. Mechanisms of
immune tolerance induction through the thymic expression
of a peripheral tissue-specific protein. Int. Immunol. 1995;
7(5): 715–725.
3. Hosono M., Kurozumi M., Inaba M. et al. Neonatal tolerance
induction in the thymus to MHC-class II-associated
antigens. IV. Significance of intrathymic chimerism of
blood-born Ia+ cells in Mls tolerance. Cell Immunol. 1991;
136(2): 373–387.
4. Jenkinson E. J., Jhittay P., Kingston R., Owen J. J. T. Studies
of the role of the thymic environment in the induction of
tolerance to MHC antigens. Transplantation 1985; 39:
331–333.
5. Matzinger P., Guerder S. Does T-cell tolerance require a
dedicated antigen-presenting cell? Nature 1989; 339: 74–76.
6. Steinman R. M. The dendritic cell system and its role in
immunogenicity. Annu. Rev. Immunol. 1991; 9: 271–296.
7. von Gaudecker B. Functional histology of the human
thymus. Anat. Embryol. 1991; 183(1): 1–15.
8. Bearman R. M., Bensch K. G., Levine G. D. The normal
human thymic vasculature: an ultrastructural study. Anat.
Rec. 1975; 183(4): 485–497.
9. Ranga V., Ispas A. T., Chirulescu A. R. Elements of structure
and ultrastructure of the blood-thymus barrier in ACTH
involuted thymus. Acta Anat. 1982; 111(3): 177–189.
10. Nieuwenhuis P., Stet R. J., Wagenaar J. P., Wubbena A. S.,
Kampinga J., Karrenbeld A. The transcapsular route: a new
way for (self-) antigens to by-pass the blood-thymus barrier?
Immunol. Today 1988; 9(12): 372–375.
11. Oluwole S. F., Jin M. X., Chowdhury N. C., Ohajekwe O. A.
Effectiveness of intrathymic inoculation of soluble antigens
in the induction of specific unresponsiveness to rat islet
allografts without transient recipient immunosuppression.
Transplantation 1994; 58(10): 1077–1081.
12. Hosono M., Kurozumi M., Inaba M. et al. Neonatal tolerance
induction in the thymus to MHC-class II-associated
antigens. IV. Significance of intrathymic chimerism of
blood-born Ia+ cells in Mls tolerance. Cell Immunol. 1991;
136(2): 373–387.
13. Hosono M., Hosokawa T., Kina T., Katsura Y. Neonatal
tolerance induction in the thymus to MHC-class
II-associated antigens. III. Significance of hemopoietic stem
cells for induction and maintenance of Mls tolerance by
continuous supply of tolerance-inducing nonlymphocytes.
Cell Immunol. 1987; 108(1): 162–174.
14. Wilson D. B., Wilson D. H., Schroder K. Acquired thymic
tolerance and experimental allergic encephalomyelitis in the
rat. I. Parameters and analysis of possible mechanisms. Eur.
J. Immunol. 1998; 28(9): 2770–2779.
15. Ellison G., Waksman B. Role of the thymus in tolerance. IX.
Inhibition of experimental autoallergic encephalomyelitis
by intrathymic injection of encephalitogen. Immunology
1970; 105: 322–356.
16. Najman S., Bubanovic I., Stamenkovic S., Petrovic S.,
Stankovic Z. Effects of inoculating adherent thymic cells of a
Medical Hypotheses (2003) 60(4), 520–524
524 Bubanovic
donor into the thymus of a recipient on the survival of the
skin graft. Acta Medica. Medianae. 1990; 1: 79–88 (Non
English article).
17. Garrovillo M., Ali A., Oluwole S. F. Indirect allorecognition
in acquired thymic tolerance: induction of donor-specific
tolerance to rat cardiac allografts by allopeptide-pulsed host
dendritic cells. Transplantation 1999; 68(12):
1827–1834.
18. Oluwole S. F., Chowdhury N. C., Jin M. X., Hardy M. A.
Induction of transplantation tolerance to rat cardiac
allografts by intrathymic inoculation of allogeneic soluble
peptides. Transplantation 1993; 56(6): 1523–1527.
19. Carp H., Torchinsky A., Toder V. Maternal alloimmune
stimulation: Variable actions. In: S. K. Gupta (ed).
Reproductive Immunology. Delhi: Norsa Publishing House,
1999: 205–217.
20. King A., Gardner L., Loke Y. W. Human decidual leucocytes
do not proliferate in response to either extravillous
trophoblast or allogeneic peripheral blood lymphocytes.
J. Reprod. Immunol. 1996; 30: 67–74.
21. Maejima M., Fujii T., Kozuma S., Okai T., Shibita Y.,
Taketani Y. Presence of HLA-G expressing cells modulates
the ability of peripheral blood mononuclear cells to release
cytokines. Am. J. Reprod. Immunol. 1997; 38: 79–82.
22. Ljunggren G., Karre K. In search of the ‘missing self’-MHC
molecules and NK cell recognition. Immunol. Today 1990;
11: 237–241.
23. Hill A. J. Cytokines in early pregnancy success and failure.
In: Cytokines in Human Reproduction. Wiley-Liss; 2000. p.
161–169.
24. Beutler B., Von Haffel C. Unraveling function in the TNF
ligand and receptor families. Science 1994; 264: 667–668.
25. Lin H., Mossman T. R., Guilbert L., Tuntipopipat S.,
Wegmann T. G. Sythesis of T helper-2 type cytokines at the
maternal–fetal interface. J. Immunol. 1993; 151: 4562–4573.
26. Wegmann T. G., Lin H., Guilbert L., Mosman T. R.
Bidirectional cytokine interactions in the maternal–fetal
relationship. Is successful pregnancy a Th2 phenomenon?
Immunol. Today 1993; 14: 353–356.
Medical Hypotheses (2003) 60(4), 520–524
27. Tangri S., Raghupathy R. Expression of cytokine in placenta
of mice undergoing immunologically mediated spontaneous
fetal resorption. Biol. Reprod. 1993; 49: 850–856.
28. Tangri S., Wegamnn T. G., Raghupathy R. Maternal
anti-placental reactivity in natural immunologically
mediated fetal resorption. Biol. Reprod. 1993; 49: 850–856.
29. Ho H. N. Activation status of T and NK cells in the
endometrium throughout menstrual cycle and normal and
abnormal early pregnancy. Hum. Immunol. 1996; 49:
130–135.
30. Hayakawa S., Karasaki-Suzuki M., Itoh T. et al. Effects of
paternal lymphocyte immunization on peripheral Th1 /Th2
balance and TCR Vb and Vc repertoire usage of patients with
recurrent spontaneous abortions. Am. J. Reprod. Immunol.
2000; 43(2): 107–115.
31. Kwak J. Y., Gilman-Sachs A., Moretti M., Beaman K. D., Beer
A. E. Natural killer cell cytotoxicity and paternal lymphocyte
immunization in women with recurrent spontaneous
abortions. Am. J. Reprod. Immunol. 1998; 40(5): 352–358.
32. Katz I., Fisch B., Amit S., Ovadia J., Tadir Y. Cutaneous
graft-versus-host-like reaction after paternal lymphocyte
immunization for prevention of recurrent abortion. Fertil.
Steril. 1992; 57(4): 927–929.
33. Bux J., Westphal E., de Sousa F., Mueller-Eckhardt G.,
Mueller-Eckhardt C. Alloimmune neonatal neutropenia is a
potential side effect of immunization with leukocytes in
women with recurrent spontaneous abortions. J. Reprod.
Immunol. 1992; 22(3): 299–302.
34. Tanaka T., Umesaki N., Nishio J. et al. Neonatal
thrombocytopenia induced by maternal anti-HLA
antibodies: a potential side effect of allogeneic leukocyte
immunization for unexplained recurrent aborters. J. Reprod.
Immunol. 2000; 46(1): 51–57.
35. Kamenov B. Dysfunction of the immune system and chronic
diseases in children born from mothers with previous
spontaneous abortions. In: 14th Annual Conference on
Clinical Immunology and 5th International Symposium on
Clinical Immunology, Washington, USA, Clinical
Immunology, 1999: Abstract: 469.
ª 2003 Elsevier Science Ltd. All rights reserved.